Imaging lipid order changes in endosome membranes of live cells by using a Nile Red-based membrane probe

Abstract

During maturation of endosomes, in addition to the decrease in their internal pH and the modifications of their proteins, changes are thought to occur at the level of their lipid membranes. In the present work, we used the recently developed environment-sensitive membrane probe NR12S to monitor in living cells the lipid order changes that accompany the maturation of endosomes. Internalization studies in HeLa cells using two-photon fluorescence microscopy in the presence of endocytosis markers and inhibitors show that the probe is endocytosed through different energy-dependent pathways. While marginal changes in the probe colour occur during the initial steps of endocytosis, dramatic changes in colour are observed in the membranes of late endosomes and lysosomes. This remarkable colour change, which takes place 2 h after initial binding of NR12S to cell plasma membranes, suggests a loss of lipid ordered phase during endosome maturation. This change of lipid phase likely results from the decrease in the cholesterol content and the hydrolysis of sphingomyelin occurring in the membranes of late endosomes and lysosomes. In comparison to the common endocytosis marker FM4-64, NR12S is many-fold brighter, it can monitor in situ changes in the lipid organization of endosome membranes and its fluorescence at the plasma membrane can be selectively switched off by sodium dithionite. The present work proposes thus a new powerful tool for endocytosis research that enables monitoring changes in the lipid composition of endosome membranes.